Morphological and Phylogenetic Characterization of Three Novel Thaxterogaster (Cortinariaceae) Species from China with an Emphasis on Their Subtropical Distribution

Three new phlegmaciod species of Thaxterogaster, T. borealicremeolinus, T. rufopurpureus, and T. sinopurpurascens spp. nov., from subtropical China were described based on their morphological characteristics and molecular data. Thaxterogaster borealicremeolinus belongs to the sect. Cremeolinae and differs from the other species in this section in its larger basidiospores and its habitat in the Northern Hemisphere associated with Quercus sp. trees. Thaxterogaster rufopurpureus and T. sinopurpurascens belong to sect. Purpurascentes, in which T. rufopurpureus is characterized by a pileus with a reddish-brown coloration when mature and a clavate stipe, while T. sinopurpurascens is characterized by a violet basidiomata, except for a greyish orange to brown pileus, the distinctly marginate bulb of its stipe, and its distribution in subtropical China. The phylogenetic analyses were performed based on nrITS, and detailed descriptions of the new species are provided herein.


Introduction
Thaxterogaster Singer was proposed as a sequestrate genus in Gasteromycetes Fr., Secotiaceae Tul.& C. Tul.[1].Later, this genus was changed to Cortinariaceae R. Heim [2].Thaxterogaster was used by taxonomists as an independent genus until the early part of this century when molecular phylogenetic studies showed that this genus was nested within Cortinarius (Pers.)Gray and was thus proposed as a synonym of Cortinarius [3].Recently, the genus Cortinarius was split into ten genera based on multi-gene sequence and genomic data; Thaxterogaster was emendated and, as a genus, separated from other genera of Cortinariaceae, which followed the Shenzhen Code (Art.11.4 of the Code) [4].
The currently accepted concept of the genus Thaxterogaster is a morphologically diverged group of fungi comprising the phlegmacioid and myxacioid agaric species that were previously classified within Cortinarius and some sequestrate species in early genera like Thaxterogaster and Gigasperma E. Horak [4].Currently, the genus Thaxterogaster is recognized as bihemispherical, with more than 170 species described all over the world [5], and it includes six subgenera and 23 sections [4].
Currently, there are only 22 known Thaxterogaster species in China, but before Liimatainen et al. emendated this genus, these species were recorded in the genus Cortinarius s.l.[8][9][10][11].In the past, the research on Cortinariaceae species focused on the temperate zones of China and its Tibetan Plateau, publishing a series of new species [12][13][14][15] but paying less attention to Cortinariaceae species in the subtropical and tropical regions of China.Recently, we collected some specimens of Cortinariaceae from subtropical China, some of which are new and rare species.Here, we describe three Thaxterogaster species collected from subtropical China and establish them as new to science based on morphological and ecological evidence and phylogenetic results.

Specimens and Morphological Description
Specimens included in this study were collected in subtropical China in spring and early summer.Basidiomata were photographed in the field and dried in an oven at about 50 • C. All specimens were deposited in the Herbarium Mycologicum, Academiae Sinicae, Institute of Microbiology, Beijing, China (HMAS).
The macroscopic characteristics were described based on fresh basidiomata.The basidiospores, basidia, sterile cells, and pileipellis were observed in a 5% potassium hydroxide water solution, Congo Red, and Melzer's reagent under a light microscope.Thirty mature basidiospores were measured per specimen, and the Q (L/W ratio) values were calculated for all basidiospores.X av. and Q av.indicate the average values of basidiospores in every specimen.Twenty basidia and sterile cells per specimen were measured from the pieces of lamellae.The pileipellis structures were studied from radial sections situated halfway from the pileus center.The photos of basidiospores and pileipellis were taken with an EMCOMOS Camera HY-2000W.

Molecular Phylogeny
The genomic DNA was extracted from the dried specimens using the standard cetyltrimethylammonium bromide (CTAB)-chloroform method with a few modifications [16,17].The primers ITS1F and ITS4 were used to amplify the ITS region [18,19].The PCR procedures are as follows: initial denaturation at 95 • C for 5 min, followed by 35 cycles at 95 • C for 30 s, 48 • C for 30 s, and 72 • C for 1 min and a final extension of 72 • C for 10 min.Sequencing was performed by Beijing Tsingke Biotech (Beijing, China) Co., Ltd.All the newly generated sequences were submitted to GenBank, and BLASTn was run in NCBI to select the closely related species for the phylogenetic analyses.We also selected sequences from the other two sections, i.e., sect.Multiformes Niskanen & Liimat.and sect.Riederorum Niskanen & Liimat., for phylogeny reconstruction because they are also members of Thaxterogaster [4].Two Phlegmacium (Fr.) Wünsche species, P. cyanites (Fr.)M.M. Moser and P. boreicyanites (Kytöv., Liimat., Niskanen & A.F.S. Taylor) Niskanen & Liimat., were chosen as an out-group.All sequences used in the phylogenetic analyses are shown in Table 1.
ITS dataset used for phylogenetic analyses was aligned and manually adjusted using BioEdit 7.1.3.0 [20].Phylogenetic analyses were performed using Bayesian Inference (BI) and Maximum Likelihood (ML) methods.The best model (GTR + I + G) for BI analysis was chosen according to the Akaike information criterion (AIC) using the software MrModeltest 2.3 [21].Bayesian analysis was performed with MrBayes 3.2.6 [22].Two independent analyses of two parallel runs and four chains were carried out for 10,000,000 generations, sampling every 1000 generations.The first 25% of the trees were discarded as burn-in.The ML analysis was implemented using RAxML 8.2.12 in raxmlGUI with a rapid bootstrapping algorithm of 1000 replicates [23,24].Default parameters of the GTRGAMMA model were used in the ML analysis.Newly generated sequences were marked in bold.

Molecular Phylogeny
A total of 65 sequences, including 41 from type materials, were used in the phylogenetic analyses, representing 44 species and one undescribed species.The BI and ML trees showed similar topologies, and the BI tree was selected as a representative example (Figure 1).The Bayesian posterior probabilities (BPP) ≥ 0.80 and ML bootstrap values (ML) ≥ 60% are shown on the branches (BPP/ML).Thaxterogaster species were clustered into four clades in the phylogenetic tree, representing four instinct sections, i.e., sect.Cremeolinae (Soop) Niskanen & Liimat., sect.Multiformes, sect.Purpurascentes (Kühner & Romagn.ex Brandrud & Melot) Niskanen & Liimat., and sect.Riederorum, respectively.The sect.Purpurascentes clade (BPP/ML = 1.00/86%) included fourteen known species, two novel species, and one undescribed species.Thaxterogaster rufopurpureus nom.prov.could be separated from all the other species in this section.For another new species, T. sinopurpurascens nom.prov., our five collections together with HKAS 12252 (recorded as T. purpurascens) were clustered into a distinct lineage with strong statistical support (BPP/ML = 0.99/93%), which was found to be closely related to T. purpurascens and T. indopurpurascens    Description: Pileus 3-6.7 cm in diam., hemispherical at first, later plano-convex, viscid, yellowish-brown to reddish-brown, the margin persistently wavy.Lamellae emarginated, moderately crowded, whitish at first, greyish-brown when mature, edges even.Stipe 2.5-6.5 cm long, 0.7-1.1 cm thick at the apex, cylindrical with an expanded base (somewhat marginate bulb), up to 1.6 cm, whitish, surface with yellowish fibrils, basal mycelium white.Universal veil yellowish.Context thick, white.
Ecology and distribution: Gregarious in subtropical mixed forests.Known to inhabit Zhejiang Province, China.It may also distribute in Yunnan Province, China, and Japan, associated with Monotropa hypopithys and Pseudotsuga japonica according to the sequence information from GenBank.
Notes: Thaxterogaster borealicremeolinus is characterized by a yellowish-to-brownish, viscid pileus with a persistently wavy margin, whitish lamellae and stipe, large basidiospores, and a Northern Hemisphere distribution.Phylogenetically, T. borealicremeolinus is clustered with the species of sect.Cremeolinae and has formed a sister relationship with T. dulciorum, but the latter is only distributed in New Zealand, associated with Nothofagus, and the basidiospores are small, being <8 µm long [25].Molecularly, the most closely related species are T. cremeorufus (94.16% similarity in ITS) and T. nebulobrunneus (94.15% similarity in ITS), in which T. cremeorufus is also distributed in the Southern Hemisphere, and associated with Myrtaceae trees and with smaller basidiospores < 10 µm long [26], while T. nebulobrunneus is a distinctly sequestrate species occurring in sub-alpine grassy woodland among Eucalyptus from Australia [27].
Ecology and distribution: Gregarious in subtropical mixed forests.Known to inhabit Zhejiang Province, China.
Notes: Thaxterogaster rufopurpureus is characterized by a greyish-purple to reddishbrown pileus of viscid, turning deeper purple when bruised at any part, moderately verrucose basidiospores, and a subtropical habitat.In phylogenetic terms, T. rufopurpureus formed a separate lineage in the sect.Purpurascentes, but its relationship with other species in this section is unclear.Molecularly, the most closely related species is T. argyrionus  Notes: Thaxterogaster rufopurpureus is characterized by a greyish-purple to reddi brown pileus of viscid, turning deeper purple when bruised at any part, moderately v rucose basidiospores, and a subtropical habitat.In phylogenetic terms, T. rufopurpur formed a separate lineage in the sect.Purpurascentes, but its relationship with other spec in this section is unclear.Molecularly, the most closely related species is T. argyrio   Description: Pileus 2-7 cm in diam., hemispherical at first, later plano-convex, nately fibrillose, glutinous, greyish-orange to brown, usually with a violet tinge, especia when young, with a paler margin, surface turning deeper purple when bruised.Lame emarginated, moderately crowded, violet at first, brownish-violet when mature, ed sometimes uneven, turning deeper purple when bruised.Stipe 2.2-6 cm long, 0.4-1 Fungal Names: FN 571637.Etymology: The epithet 'sinopurpurascens' refers to the affinity for T. purpuracsens and the type locality in China.
Description: Pileus 2-7 cm in diam., hemispherical at first, later plano-convex, innately fibrillose, glutinous, greyish-orange to brown, usually with a violet tinge, especially when young, with a paler margin, surface turning deeper purple when bruised.Lamellae emarginated, moderately crowded, violet at first, brownish-violet when mature, edges sometimes uneven, turning deeper purple when bruised.Stipe 2.2-6 cm long, 0.4-1 cm thick at the apex, marginate bulb at the base, ranging up to 2 cm, violet, surface with violet fibrils, turn deeper purple when bruised, basal mycelium purple tinge.Universal veil violet.Context violet at first, later whitish with a violet tinge.

Discussion
In sect.Cremeolinae, only five known species were reported across the globe before this study, and all the species are distributed in the Southern Hemisphere [31].So, T. borealicremeolinus is the first Northern-Hemisphere-distributed species in this section.Two mycorrhizal sequences, in which JQ396472 is from Monotropa hypopithys in Yunnan, China, and AB807927 is from Pseudotsuga japonica in Japan, which were clustered together with our specimen, were treated as new species.Thaxterogaster is usually associated with species of Fagaceae, Betulaceae, Tilia, and Pinaceae in the Northern Hemisphere and associated with Nothofagaceae in the Southern Hemisphere [4].The sequence JQ396472 shows that some species of Monotropoideae, a group of non-autotrophic plants, may also be a host plant of Thaxterogaster species [32].
In sect.Purpurascentes, two new species formed separate clades in the phylogenetic trees, and their morphological and ecological characteristics also approved their novelty.One specimen from Yunnan province, China (i.e., HKAS122529), was identified as T. purpurascens; here, we treated it as misidentified due to the ITS sequence clustered with T. sinopurpurascens and as distinct from the European T. purpurascens.The latter has been widely reported in China in the past, but so far, we have not found any exact specimens to confirm its Chinese distribution.
Based on our work in recent years, we have confirmed twelve known Thaxterogaster species distributed in China [10,11].But there are still ten Thaxterogaster species reported to inhabit China in previous studies that lacked molecular data, and some of them also did not provide morphological features.The lack of intensive research on Thaxterogaster has led to a deficit of knowledge of the true species diversity of this genus in China.
Here, we present the first research article focusing on the genus Thaxterogaster from China.Moreover, the basal lineage of Cortinariaceae [4,31] also shows the evolutionary importance of Thasterogaster in Cortinariaceae.So, this genus in China needs further study in the future.

Figure 1 .
Figure 1.BI tree inferred from ITS sequences.The tree is rooted in Phlegmacium species.Bayesian posterior probabilities (≥0.80) and ML bootstrap values (≥60%) are shown on each branch (BPP/ML).New species are indicated by red font.

Figure 3 .
J. Fungi 2023, 9, x FOR PEER REVIEW 7 o related species are T. cremeorufus (94.16% similarity in ITS) and T. nebulobrunneus (94.1 similarity in ITS), in which T. cremeorufus is also distributed in the Southern Hemisphe and associated with Myrtaceae trees and with smaller basidiospores < 10 µm long [2 while T. nebulobrunneus is a distinctly sequestrate species occurring in sub-alpine gra woodland among Eucalyptus from Australia [27].Thaxterogaster rufopurpureus M.L. Xie, Yi Li & W.F. Lin, sp.nov.

Table 1 .
Voucher information and their GenBank accession numbers for ITS sequences.